MARINE ECOLOGY PROGRESS SERIESMar Ecol Prog Ser

Vol. 276: 147–159, 2004 Published August 2

INTRODUCTION

The introduction of alien species has been recognizedas a major threat to marine ecosystems, with a set ofecological and evolutionary consequences from species

to ecosystem levels (Grosholz 2002). In this respect,introduced species are considered one of the mostserious threats to the conservation of natural biodiver-sity (Coblentz 1990, Cohen & Carlton 1998, Mooney &Cleland 2001, Palumbi 2001, Shea & Chesson 2002)

© Inter-Research 2004 · www.int-res.com*Email: g.bachelet@epoc.u-bordeaux1.fr

Invasion of the eastern Bay of Biscay by the nassariidgastropod Cyclope neritea: origin and effects on

resident fauna

Guy Bachelet1,*, Benoît Simon-Bouhet2, 3, Céline Desclaux1,Pascale Garcia-Meunier2, Guillaume Mairesse1, Xavier de Montaudouin1,

Hélène Raigné1, Karine Randriambao1, Pierre-Guy Sauriau4, Frédérique Viard3

1Laboratoire d’Océanographie Biologique, UMR 5805 CNRS-Université Bordeaux 1, Station Marine d’Arcachon, 2 rue du Professeur Jolyet, 33120 Arcachon, France

2Laboratoire de Biologie et Environnement Marins, EA 3168, Université de La Rochelle, 17000 La Rochelle, France3Evolution et Génétique des Populations Marines, UMR 7127 CNRS, Station Biologique de Roscoff,

BP 74, 29682 Roscoff cedex, France4Centre de Recherche sur les Ecosystèmes Marins et Aquacoles, UMR 10 CNRS-IFREMER, Place du Séminaire,

BP 5, 17137 L’Houmeau, France

ABSTRACT: The distribution area of the nassariid gastropod Cyclope neritea (L.) includes theMediterranean and the Black Sea, as well as the Atlantic coasts of the southern Iberian Peninsula. Thespecies has spread north to the eastern Bay of Biscay (Arcachon Bay in 1976, Marennes-Oléron areaand Morbihan Gulf in 1983–84). This spread might be explained either by (1) a natural spreadfavoured by environmental changes (e.g. an increase of temperature) or (2) a sudden range expansiondue to the introduction of individuals from distant native populations. Molecular genetic analysesbased on mitochondrial markers suggest that the present C. neritea population in Arcachon Bay hasbeen introduced, probably unintentionally with oyster transfers, from several source populations,genetically similar to the populations analysed in this study, i.e. those in the western Mediterraneanand in south Portugal. Within its new distribution area, C. neritea could potentially compete with theautochthonous nassariid Nassarius reticulatus, both species being scavengers. Although C. neriteatends to occur mainly in relatively clean sands in the intertidal and N. reticulatus in subtidal, organicrich sediments, the habitats of both species partially overlap. Laboratory experiments showed that instill water conditions C. neritea was more active and reached carrion faster than N. reticulatus, therebyhaving a competitive advantage over the latter; flow conditions ( ~1 cm s–1) appeared to stimulate theactivity of N. reticulatus. Analysis of parasite load in both species in Arcachon Bay indicated thatN. reticulatus was more heavily parasitized by digenean trematodes than C. neritea. This suggests thatthe spread and population dynamics of C. neritea along the French Atlantic coast has been favouredby the lack of parasites. Altogether, recurrent introduction, competitive ability and lack of heavyparasitic load might explain the successful settlement of C. neritea along the French Atlantic coast.

KEY WORDS: Introduced species · Scavenging gastropods · Population genetics · Interspecificcompetition · Behaviour · Parasites

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Mar Ecol Prog Ser 276: 147–159, 2004

and are identified as the second cause (after habitatdestruction by human activities) of the loss of biologi-cal diversity (Vitousek et al. 1997, Wilcove et al. 1998).As recently emphasized by Ruiz et al. (2000), multipleapproaches, although rare, are required to understandthe patterns and processes of marine biological inva-sions. For example, to identify the proximate factors forthe success or the failure of invasion, studies of life his-tory traits, species interactions (including the conse-quences on resident species) and the genetic architec-ture of invasive populations are complementary(Parker et al. 1999, Ruiz et al. 2000, Sakai et al. 2001,Grosholz 2002, Lee 2002).

Dukes & Mooney (1999), Carlton (2000) and Sta-chowicz et al. (2002) have pointed out the likelyrelationships between global and climate change andthe success of biological invasions. In this context, thenassariid gastropod Cyclope neritea (L., 1758) is aninteresting model species. This gastropod is 1 of the 87marine and brackish water species that are known orsuspected to have been accidentally introduced to theAtlantic and Channel coasts of France (Goulletquer etal. 2002). C. neritea is native to the Mediterranean andBlack Seas and to the Atlantic coasts of southern Spainand Portugal as far north as Setubal (Hidalgo 1917,Nobre 1931). Up to the early 1970s, discontinuous pop-ulations were also recorded in the north of Spain andon the French Basque coast (Kisch 1950, Morton 1960;see also Sauriau 1991 for a thorough analysis of occur-rence records of C. neritea on the Atlantic coasts). NewC. neritea populations appeared on the French At-lantic coasts during the 1970s and 1980s: (1) in 1976 inArcachon Bay (Bachelet et al. 1980), (2) in 1983–84 fur-ther north, in the Marennes-Oléron Bay (Pigeot 1988,Sauriau 1989), at the Isle of Ré (Tardy et al. 1985), andin the Gulf of Morbihan (Le Roux et al. 1988). ArcachonBay and the Gulf of Morbihan are located approxi-mately 150 km and >500 km, respectively, north of theearly 1970s range of the species. It should be notedthat, in all these newly colonized areas, C. neritea pop-ulations are now self-reproducing and abundant, withdensities >100 ind m–2 (Tardy et al. 1985, Bachelet etal. 1990, Le Roux 1994, Afli & Chenier 2002). A largeC. neritea population was also observed in 2003 in theBay of Morlaix, English Channel (B. Simon-Bouhet & F.Viard, pers. obs.).

Two non-exclusive hypotheses can be put forward toexplain the present distribution of Cyclope neriteaalong the French Atlantic coasts: (1) a natural rangeexpansion towards the north due to environmental(e.g. climatic) changes, and/or (2) accidental introduc-tions related to shellfish culture (e.g. oyster transfersbetween the Mediterranean and the Bay of Biscay).As suggested by Sauriau (1991), both processes mayhave been responsible, since the aforementioned new

populations of C. neritea have potentially benefitedfrom a slight warming of coastal waters during theperiod 1970 to 1990. However, when a species is intro-duced close to its natural distribution area, it is difficultto discriminate both processes using field surveyonly. We addressed this issue by sequencing a mito-chondrial DNA gene fragment (the Cytochrome C Oxi-dase I, COI), which is known to be a powerful tool indetermining the origin and history of new populations(Sakai et al. 2001) and can trace back historicalprocesses at within-species level (Avise et al. 1987,Avise 1991).

Competitive interactions between native and intro-duced species, leading to fitness reduction in nativepopulations, have been experimentally demonstratedin marine species and found to be a major determinantof the success of an invasion (see e.g. reviews byCarlton 1992, Ruiz et al. 1999, Grosholz 2002). In Euro-pean coastal waters an autochthonous species withwhich Cyclope neritea might compete is the netteddogwhelk Nassarius reticulatus (L.), as both speciesbelong to the same family and feed as scavengers.We made field observations to determine if bothspecies when living in sympatry occupy a similarhabitat, and performed laboratory experiments todetermine if there was competition for food betweenthe 2 nassariids.

It has also been suggested that parasite release is im-portant in allowing an introduced species to becomeinvasive (Calvo-Ugarteburu & McQuaid 1998b). Par-asitic infection in introduced species is mainly limitedby 2 fundamental principles (Torchin et al. 2002):(1) the intensity and prevalence of infection tend to in-crease with host population density, which suggeststhat in a recently introduced host-parasite system, hostdensity will often be too low for the parasite to estab-lish and (2) parasites, in particular digenean trema-todes that exhibit high prevalence in molluscs (Lauck-ner 1980), often have a complex life-cycle, including 2or more host species. Consequently, many introducedspecies lack most or all of their native natural parasites(Calvo-Ugarteburu & McQuaid 1998a, Torchin et al.2001), which is advantageous when in potentialcompetition with native species. In the present workwe compared the parasite prevalence and richnessbetween Cyclope neritea and Nassarius reticulatus.The disadvantage in being parasitized was assessedthrough a survival experiment.

Based on an integrative and pluridisciplinary ap-proach, the aims of this study are therefore to giveinsights about the history of the introduction, togetherwith the identification of biological factors that mayhave promoted the settlement and then the mainte-nance of an exotic marine species, recently introducedalong the European North Atlantic coast.

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Bachelet et al.: Invasion by Cyclope neritea

MATERIALS AND METHODS

Study species and area. Cyclope neritea is a smallmollusc (~15 mm maximum shell width) which livesin shallow and intertidal habitats, predominantly incalm waters, where it usually lies buried a few mmbelow, or crawls at, the sediment surface. Like mostmembers of the family Nassariidae, it is predomi-nantly a scavenger, feeding on carrion, especiallydead bivalves (Morton 1960, Britton & Morton 1994),though it may also behave as a deposit feeder(Southward et al. 1997). C. neritea has no dispersivelarval stage: the embryos undergo direct developmentduring 2 to 5 wk within egg capsules (each capsuleusually containing a single embryo) deposited onhard substrata such as cockle shells, then hatch asbenthic juveniles (Kisch 1950, Gomoiu 1964, Massé etal. 1978, Boulhic & Tardy 1986, D’Asaro 1993). Thefirst C. neritea population observed outside its com-mon native range (Mediterranean, Black Sea andAtlantic coasts of the Iberian Peninsula) was inArcachon Bay, where it has been monitored since1976. We focused on this particular population todevelop several parallel studies (population genetics,habitat, competition and parasitic load).

Sampling, DNA extraction, amplification andsequencing. Adult specimens of Cyclope neriteawere collected from 3 intertidal locations (Table 1):(1) Faro (S Portugal) and (2) Thau Lagoon (FrenchMediterranean), which are both located within thenatural range of the species; these populations mayhave contributed to the settlement of C. neritea inFrance; and (3) Arcachon Bay (site ‘Arguin’), wherethe first new population of C. neritea on the FrenchAtlantic to the north of the Basque coast wasrecorded. The specimens were attracted with a deadfish, collected, removed from their shell and storedindividually in 95% ethanol before DNA extraction.Total DNA was extracted from <15 mg of foot muscleusing DNeasyTM Tissue Kit according to the manufac-turer’s protocol (Quiagen). From preliminary sequen-ces carried out with the universal primers originallydefined by Folmer et al. (1994) (i.e. LCO1491 andHCO2198) to amplify the COI gene, we designed a

new set of internal primers more specific to C. neritea :Cy2 5’-GTTAAAATTTCGATCTGTTA-3’ (forward)and CyR 5’-GGATTAGTTGGTACAGC-3’ (reverse).The amplifications were performed using theseprimers in 50 µl reactions containing 5 to 50 ng DNA,0.5 µM of each primer, 0.2 mM dNTPs, 1.5 mMMgCl2, 1× Reaction Buffer (containing 50 mM TrisHCl pH 8.8 at 25°C, 200 mM (NH4)2SO4, ABgene)and 1.25 U Red Hot® DNA Polymerase (ABgene). AMJResearch PTC 100 Thermal Cycler was used witha cycling profile as follows: initial denaturation stepat 93°C (3 min) followed by 40 cycles at 93°C (30 s),53°C (30 s), 72°C (60 s) and a final extension step at72°C (10 min). Double stranded PCR products werecleaned using MultiScreen-PCR MANU03010 plates(Millipore) and cycle-sequenced using ABI PRISM®BigDye™ Terminators v3.0 Cycle Sequencing Kit(Heiner et al. 1998) following the manufacturer’s pro-tocol (Applied Biosystems). Unincorporated BigDyeterminators were removed using MultiScreenMAHVN4510 plates (Millipore). In order to minimizesequencing errors, both strands were sequenced foreach individual using an ABI PRISM® 3100 Auto-mated DNA Sequencer (Perkin-Elmer Applied Bio-systems). Sequence data were aligned usingClustalW (Thompson et al. 1994) and ambiguitieswere checked comparing each sequence with itscomplementary fragment using BioEdit (Hall 1999).

Sequence analysis. A haplotype is defined by aunique stretch of nucleotides. Haplotype diversity He

(Nei 1987) and average pairwise difference K (i.e.average number of nucleotide differences betweenpairs of sequences) (Tajima 1983) were calculated foreach population using the software DnaSP (Rozas &Rozas 1999). Haplotype frequencies and pairwise FST

values (calculated from frequency values; Weir &Cockerham 1984) were estimated using Arlequinv.2.0 (Schneider et al. 2001). Using a permutationprocedure as implemented in Arlequin v.2.0, weassessed the statistical significance of pairwise FST

values under the null hypothesis of no differencebetween populations.

For polymorphic populations, the mismatch distrib-ution (i.e. the distribution of the observed number of

pairwise nucleotide site differences) wascomputed in DnaSP, in order to examinethe occurrence of mixing of evolutionarydivergent haplotype groups at the popu-lation level. This analysis was used to putforward hypotheses about the number ofgenetically differentiated populations atthe origin of the establishment of a givenpopulation.

Finally, phylogenetic analyses wereperformed with the software Network

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Table 1. Cyclope neritea. Sampling sites (geographic co-ordinates) and mole-cular diversity of populations. The number of individuals sequenced (N), thenumber of haplotypes (H), the haplotype diversity (He) and the average pairwisedifferences (K) are given for each studied population and for the entire data set

Latitude Longitude N H He K

Thau Lagoon 43° 27’ N 3° 38’ E 29 1 0.000 0.000Faro (Rio Formosa) 37° 00’ N 7° 58’ W 31 1 0.000 0.000Arcachon Bay (Arguin) 44° 35’ N 1° 14’ W 33 5 0.725 6.576Total - - 93 5 0.602 6.344

Mar Ecol Prog Ser 276: 147–159, 2004

which builds haplotypic networks from free-recombi-nant data, based on the median-joining algorithm(Bandelt et al. 1999). This process combines bothminimum spanning trees and maximum parsimonyapproaches. Such networks show the evolutionaryrelationships (based on mutational events) betweenthe different haplotypes observed over the wholedata set.

Habitat preferences. The habitat, in terms of bathy-metric distribution and sediment properties, ofCyclope neritea and Nassarius reticulatus wasassessed in Arcachon Bay in March 2002. In thismacrotidal lagoon, 5 bathymetric transects wereperformed, with 4 to 10 sampling stations on each tran-sect. Stations were distributed over a tidal range from–8.8 to +3.1 m. At each station, a trap (diameter 26 cm),made of a plastic bag with mesh openings of 4 mm onthe bottom (to prevent loss of nassariids when raised tothe surface) and 15 mm on the sides (to allow nassariidsto enter the trap), was immersed at high tide anddeposited on the sea bottom. A sampling unit wasdefined as a trap containing 20 g fresh weight of bait(dead Crepidula fornicata) immersed for 30 min. Sevenreplicates were taken at each station. Nassariids of bothspecies were counted in each sampling unit. At eachstation, surface sediment was collected with an Ekmangrab and analysed for grain size distribution (wet siev-ing) and organic matter content (loss of weight of drysediment at 550°C during 2 h).

Experiments on interspecific competition for food.Laboratory experiments were designed to investigatethe behaviour and the chemoreception ability ofCyclope neritea and Nassarius reticulatus. Theseexperiments were performed in a long straight PVCchannel (160 × 7 × 3.5 cm) without sediment andfilled to a depth of 3 cm with seawater. No attemptwas made to control environmental parameters;however, the experiments were run during the dayunder natural lighting, and within a short period (16to 20 May) during which conditions remained largelyconstant. During the experiments, water temperatureand salinity ranges were 17.7 to 18.9°C and 24 to 28‰,respectively. Nassariids were collected in ArcachonBay and maintained without food in aquaria withrunning oxygenated seawater for 2 months prior tothe experiments. Similar-sized adult C. neritea (shellwidth = 11 to 15 mm) and N. reticulatus (shell height= 18 to 27 mm) were used. All experiments were car-ried under 2 different hydrodynamic conditions: (1) instill water, and (2) in flow conditions, where sea-waterflowed unidirectionally at a surface velocity of 0.81 to0.94 cm s–1.

The first set of experiments was conducted with-out any prey in order to observe the spontaneousbehaviour of both nassariid species. Individuals of

the 2 species (4 Cyclope neritea and 4 Nassariusreticulatus; 8 replicates) were arranged in a line atan equal distance from both ends of the channel. Thenumber of individuals moving spontaneously (in stillwater and flow conditions) over a distance of at least10 cm in 30 min was recorded.

In a second set of experiments we measured theability of both nassariid species to locate and reach aprey (a dead cockle Cerastoderma edule). Individualsof the 2 species (4 Cyclope neritea and 4 Nassariusreticulatus; 8 replicates in still water, 4 replicates inrunning seawater) were placed at one end of thechannel and allowed to feed upon the carrion whichwas placed at distances of 20, 40 or 60 cm. In flowconditions, seawater flowed unidirectionally towardsboth the carrion and the nassariids. The times atwhich each individual reached the prey wererecorded and their speed was calculated. Individualsthat did not reach the carrion within 30 min wereassigned zero.

Survey of parasite abundance. From October 2000to July 2001, sympatric populations of Nassariusreticulatus and Cyclope neritea were collectedmonthly by hand on a sandflat (‘Arguin’) in ArcachonBay. In the laboratory, 100 snails of each species weregently crushed and dissected under a stereomicro-scope. Mean prevalence (i.e. percentage of infectedindividuals; Bush et al. 1997) was calculated for eachparasite species or family.

Survival experiment in relation to parasites. Dueto the low parasite infection in Cyclope neritea (see‘Results’), the survival experiment was performedwith Nassarius reticulatus only. Survival of infectedand non-infected N. reticulatus individuals was com-pared to assess the disadvantage of being parasitizedfor the native species. A large quantity of snails wascollected at ‘Arguin’, and subsequently isolated indishes with seawater at ~24°C. Each dish wasobserved under a stereomicroscope to detect dige-nean larvae (cercariae). Nassariids that did not shedparasites were not always free of infection (Curtis &Hubbard 1990). Therefore, a higher number ofcontrol snails (×3) were kept in order to checknon-infection when individuals died. Parasitized andcontrol snails had the same size structure. A total of158 snails (36 parasitized and 122 control) were gath-ered in a large aquarium, in running seawater atambient temperature. Each individual was marked tocalculate its lifespan during the experiment (totalduration: 280 d). Snails were not fed in order toweaken them and exacerbate parasite detrimentaleffects. Although unrealistic, being starved equal-ized all gastropods at the same ‘diet’ and preventedthem from trophic infestation (de Montaudouin et al.2003).

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Bachelet et al.: Invasion by Cyclope neritea

RESULTS

Mitochondrial DNA (mtDNA) polymorphism withinand among native and introduced Cyclope neritea

populations

PCR amplification yielded a 553 base pair (bp) longfragment of the COI gene. Nineteen polymorphic siteswere found in the 3 populations, and out of the entiresample (N = 93), 5 different mtDNA haplotypes wereidentified (Table 2). Over the whole data set, 2 haplo-types were found at very high frequency (f), namely A(f = 0.462) and B (f = 0.430). Haplotypes C, D and Ewere found more rarely, with frequencies of 0.065,0.032 and 0.011, respectively. At the population level,Thau and Faro populations revealed only 1 haplotype(A and B, respectively), which was in contrast to theArcachon population where the 5 haplotypes (A to E)were found, with A and B representing about 70% ofthe total number of sequences for thispopulation (see haplotype distributionin Fig. 1). Consequently, at Arcachon,the haplotype diversity was very high(He = 0.725, Table 1) as well as theaverage pairwise difference (K =6.576, Table 1). This high value of Kwas in agreement with the mismatchdistribution from which 2 main peakswere observed, 1 of them centred onvalues of 13 to 14 nucleotide differ-ences (Fig. 2). Such values are indica-tive of mixing of groups of individualscharacterized by evolutionary diver-gent haplotypes within the Arcachonpopulation. This pattern can berelated to the evolutionary relation-ships between the observed haplo-types, as shown in the haplotypic net-work (Fig. 3). From this network, 3major clusters were identified: (1)Haplotypes A and C, (2) Haplotypes Band D, and (3) Haplotype E. Haplo-types were highly divergent amongthese groups (13 mutations between A

and B, 11 between A and E, and 13 between B and E;Fig. 3) but haplotypes were very close within eachgroup (2 mutations steps between A and C, 1 betweenB and D; Fig. 3).

151

Fig. 1. Cyclope neritea. Haplotype distribution within the 3 study populations.Total number of individuals sequenced in each population (N) and number of eachhaplotype in the population of Arcachon Bay (n) are indicated. Haplotypes A to E

are distinguished by different patterns

0.00

0.05

0.10

0.15

0.20

0.25

0.30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17Number

Fre

quen

cy

of pairwise sequence differences

Fig. 2. Cyclope neritea. Mismatch distribution for the popula-tion of Arcachon Bay. The frequency of each observed value

of pairwise difference is plotted

Positions (bp)

1 44 50 53 104 218 224 254 266 292 317 347 377 382 398 422 425 479 503A A T G G T A A T T G T G G C C C G G CB - C A A - - - C C - C A A - G T - A TC - - - - - - - - - - - - - A - - - - -D - C A A - - - C C - C A A A G T - A TE G - - A C G G C C A C - - - - - A A -

Table 2. Cyclope neritea. Definition of Haplotypes A to E found within the 3 populations of Thau, Faro and Arcachon. Positionsof the polymorphic sites are given. ‘-’ indicates that the same character is present in Haplotype A

Mar Ecol Prog Ser 276: 147–159, 2004

Pairwise FST values were all significant (p < 10–5)indicating a large and significant genetic structureamong the 3 populations. Because of the lack of com-mon haplotypes, the highest (and maximum) geneticdifferentiation was found when comparing the popula-tions of Thau and Faro (FST = 1). High genetic differen-tiation levels were also found when comparing thepopulations of Arcachon and Faro (FST = 0.58) and thepopulations of Arcachon and Thau (FST = 0.32).

Habitat of Cyclope neritea and Nassarius reticulatus

In Arcachon Bay Cyclope neritea occurred at tidallevels between –0.8 and +3.1 m, while Nassariusreticulatus was found between –5.2 m and +1.9 m(observations at the 5 study transects). As an example,Fig. 4 shows the zonation pattern along the bathymet-ric transect at ‘Arguin’, where the abundance of C.neritea was the highest at intertidal levels and that ofN. reticulatus increased with depth down to –4.0 m,with an overlap of both species distribution between–1.0 and +1.3 m. C. neritea did not occur in fine,organic-rich sediments, whereas N. reticulatus werefound in all sediment types sampled at Arcachon (Fig. 5).C. neritea did not tolerate sediment organic content >1%, whereas N. reticulatus were still abundant in sed-iment with organic content as high as 14%.

Competition for food between Cyclope neritea andNassarius reticulatus

When placed without any dead prey in the seawaterchannel, the individuals of both nassariid specieseither moved erratically or did not move at all, regard-less of hydrodynamic conditions. Nevertheless, a 2-wayANOVA indicated that Cyclope neritea moved sponta-neously in a higher proportion than Nassarius reticula-tus in both hydrodynamic conditions (p < 0.05) and thatthis spontaneous moving was greater in flow condi-tions than in still water for both species (p < 0.05)(Table 3; Fig. 6). Moreover, while the proportion of C.neritea moving without the presence of carrion wasalmost the same in still water (53.1 ± 9.9%) and in flow(59.4 ± 11.5%), a higher proportion of N. reticulatusmoved spontaneously in flow than in still water condi-tions (53.1 ± 11.0% and 18.7 ± 12.5%, respectively)

(Fig. 6) In the presence of carrion, the speed at whichindividuals of both species moved towards, andreached, food was higher in flow that in still water con-ditions (Fig. 7), due to chemical stimulus conveyed bywater. Due to heteroscedasticity that prevented usfrom performing a 3-way ANOVA between hydrody-

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Fig. 3. Cyclope neritea. Haplotypic network showing thephylogenetic relationships between the haplotypes. (S) Singlemutational events. (d) Missing haplotypes between observed

haplotypes in the data set

Nassarius reticulatusCyclope neritea

Arguin

Tidal level (m)

Num

ber

s p

er s

amp

ling

unit

Fig. 4. Cyclope neritea and Nassarius reticulatus. Mean (+SE)number of individuals of both species per sampling unit (seetext for explanations), at various tidal levels along a transect

at ‘Arguin’ (Arcachon Bay)

Source of variation df MS F

Species 1 0.848 4.330*Hydrodynamism 1 1.027 5.244*Species × Hydrodynamism 1 0.303 1.548 nsError 28 0.196

Table 3. Cyclope neritea and Nassarius reticulatus. Results ofa 2-way ANOVA, testing for the effect of species and hydro-dynamic conditions on the spontaneous moving of individualsof both species when no carrion was available for 30 min.Data were previously arcsin-transformed and homogeneity ofvariances was checked by Cochran test. ns: p > 0.05; *: p < 0.05

Bachelet et al.: Invasion by Cyclope neritea

namic conditions, species and distance to the prey, theresults were analysed separately for each hydro-dynamic condition by means of a 2-way ANOVA

(Table 4). In still water conditions, N. reticulatus tooklonger to arrive at carrion (i.e. speed was slower) thanC. neritea (p < 0.001) and the speeds of both species

153

Nassarius reticulatus

Cyclope neritea

Organic content (%)

Median grain size (µm)

Num

ber

s p

er s

amp

ling

unit

Num

ber

s p

er s

amp

ling

unit

Fig. 5. Cyclope neritea and Nassarius reticulatus. Numbers of individuals of both species per sampling unit (see text for explanations)in relation to (a) median grain size and (b) organic content of the sediment at 35 stations located along 5 transects in Arcachon Bay

Source of variation df MS F

(a) in still water conditions:Species 1 1.656 18.202***Distance 2 3.022 33.214***Species × Distance 2 0.043 0.471 nsError 281 0.091(b) in flow conditions:Species 1 36.741 9.949**Distance 2 74.702 20.228***Species × Distance 2 30.783 8.335***Error 90 3.693

Table 4. Cyclope neritea and Nassarius reticulatus. Resultsof 2-way ANOVAs, testing for the effect of species anddistance to the prey on the speed of individuals of bothspecies to reach their prey (a) in still water and (b) in flowconditions. Data were previously log(x+1) transformed andhomogeneity of variances was checked by Cochran test.

ns: p > 0.05; **: p < 0.01; ***: p < 0.001

Fig. 6. Cyclope neritea and Nassarius reticulatus. Mean (+SE)percentage of individuals of both species moving sponta-neously (i.e. without bait) over a minimum distance of 10 cmduring 30 min, in laboratory experiments with still water or

flow conditions

Nassarius reticulatusCyclope neritea

Still water Flow

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decreased the further away the carrion was placedfrom the nassariids (p < 0.001) (Table 4a; Fig. 7). Inflow conditions, the speed of both species was similarwhen nassariids were at distances of 20 and 40 cmfrom the carrion, but at 60 cm N. reticulatus arrived atprey more quickly than C. neritea (Fig. 7); as a conse-quence, the species × distance interaction was highlysignificant (p < 0.001) (Table 4b).

Parasite load in Cyclope neritea and Nassariusreticulatus

Five digenean families were found in Nassariusreticulatus from Arcachon Bay (site ‘Arguin’): Schisto-somatidae, Zoogonidae, Lepocreadiidae, Echinostom-atidae (mainly Himasthla quissetensis), Microphalli-dae, and unidentified sporocysts. Total prevalencefluctuated between 6 and 18% (Fig. 8). In contrast,Cyclope neritea was hardly parasitized, with only 1%infection by Microphallidae in November, Februaryand April (Fig. 8). At experiment completion (280 d),the percentage of parasitized N. reticulatus among sur-viving individuals was identical to that of the deadones, i.e. 50%. However, the peak of mortality wasreached earlier in parasitized snails, i.e. 100–120 d asopposed to 140–160 d in uninfected snails (Fig. 9).From the 60th day to the mortality peak, parasitizedsnails had a higher mortality rate.

DISCUSSION

Vacant habitat and/or high competitive ability, lowparasitic load, high genetic variance and climatechange are among the factors shown to be most criticalin the establishment of an exotic species (Williamson1996, Carlton 2000, Sakai et al. 2001, Grosholz 2002,Stachowicz et al. 2002). From field, empirical andexperimental studies, we analyzed patterns and pro-cesses associated with the recent settlement (less than30 yr) of Cyclope neritea populations along theAtlantic French coast. We particularly studied the firstestablished population (1976) in Arcachon Bay.

We first tackled the genetic architecture of the Arca-chon population compared with populations from theoriginal range of the species. Following accidentalintroductions of alien species, significant decreases ofpopulation genetic diversity compared to the nativerange have been regularly reported, e.g. in plants(Amsellem et al. 2000), in insects (Villablanca et al.1998, Tsutsui et al. 2000), and in marine invertebrates(Grosholz 2002). In contrast with these studies, therecently settled French Atlantic population of Cyclopeneritea (Arcachon) exhibited a far greater genetic

diversity than the populations located within the nat-ural range of the species (Thau and Faro). In contrast tothe populations of Thau and Faro, for which a total lackof polymorphism was found (only 1 haplotype per pop-ulation, He = 0), the recently settled population ofArcachon showed a very high level of polymorphism(5 haplotypes, He = 0.725).

The high genetic diversity observed at Arcachoncould either be due to an introduction from a geneti-cally diverse source population or to recurrent intro-ductions from genetically differentiated sources(Bohonak et al. 2001). Our data support the latterhypothesis. Indeed, the 5 haplotypes found in this pop-ulation were highly divergent with a mean number ofpairwise differences (K ) equal to 6.576. Together withthe mismatch analysis, this is indicative of a geneticadmixture within the Arcachon population and sug-gests the establishment of individuals originating fromgenetically distant populations. Moreover, haplotypesfound most frequently in this population were fixed inthe population of Thau (Haplotype A) and in the popu-lation of Faro (Haplotype B). This suggests that inde-pendent introductions from either these 2 sites or fromgenetically related sites occurred. In addition, Haplo-type E, genetically divergent from the other haplo-types, was not found in the 2 populations sampledwithin the natural range of the species. This could indi-

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Fig. 7. Cyclope neritea and Nassarius reticulatus. Mean (± SE)speed by which individuals of both species reached carrionplaced at 20, 40 or 60 cm in laboratory experiments with (a)

still water or (b) flow conditions

(a) Still water

(b) Flow

Distance to the prey (cm)

Sp

eed

(cm

min

–1)

Sp

eed

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min

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Bachelet et al.: Invasion by Cyclope neritea

cate that other sources (e.g. eastern Mediterranean orAdriatic Sea) may have contributed to the settlement ofthe Arcachon population. The Adriatic Sea, wherethere are high densities of Cyclope neritea, is sus-pected as being at the origin of several gastropodintroductions on the French Atlantic coasts because of

the Manila clam Tapes philippinarum shellfish trade(Goulletquer et al. 2002). Examples of possible intro-ductions are Gibbula albida, noticed in the MorbihanGulf in 1973 (Delemarre & Le Neuthiec 1995) and inArcachon Bay in 1986 (Bachelet et al. 1990), andRapana venosa, first introduced in the Black Seaduring the 1940s and the Adriatic Sea during the 1970s(Zibrowius 1991), then in southern Brittany in the late1990s (Goulletquer et al. 2002).

The recently settled Cyclope neritea population atArcachon probably has multiple origins: a Mediter-ranean source with the transfer of Haplotype A (andprobably C) and an Atlantic source with the transfer ofHaplotype B (and probably D). However, because welack other populations from the native range of thespecies, it is difficult to specify the true number ofsources for the French populations as well as the num-ber of independent introduction events (the origin ofHaplotype E is yet unknown). It would be interesting toexamine more closely the reasons for the observeddivergence between haplotypes A and B which couldbe indicative of a phylogeographic break acrossGibraltar and signal the occurrence of morphologicallyidentical cryptic species. The presence at Arcachon ofthe haplotype found in Thau Lagoon implies humanmediated dispersal mechanisms related to shellfishculture (e.g. transfers of Crassostrea gigas since the1960s between French oyster farms; Sauriau 1991).However, the presence at Arcachon of the haplotypefound in Faro could be due either to a natural rangeexpansion of the species towards the north or to intro-duction events related to human activities (e.g. importsof Crassostrea angulata from Portugal since the 1860s;Gruet & Baudet 1997). These 2 processes (natural ex-pansion and introduction) are characterized by differentevolutionary time scales (Ribera & Boudouresque 1995).Because of the low dispersal ability of C. neritea (i.e.direct development), a natural range expansiontowards the north would probably be associated with astep by step process contributing to a cline of the fre-quency of the haplotype found in Faro. Conversely,recurrent introductions are likely to leave jump disper-sal genetic signatures with random fluctuations of fre-quencies between populations. It is therefore critical toanalyze other populations located along the Atlanticcoasts of Portugal, Spain and France to discriminatethese 2 hypotheses (i.e. natural expansion or human-mediated dispersal from Portugal). The analysis ofother recently settled French populations of C. neriteawould also be useful to check for the consistency of thepattern observed in the Arcachon population.

Recent studies documented the importance of thegenetic architecture (genetic variance, response tonatural selection, etc.) of invading populations asbeing a crucial mechanism of biological invasions

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Fig. 8. Cyclope neritea and Nassarius reticulatus. Digeneanprevalence (% of infected snails) in N. reticulatus (top) and C.neritea (bottom), from October 2000 to July 2001, in Arcachon

Bay (‘Arguin’)

Fig. 9. Nassarius reticulatus. Mortality of parasitized andhealthy (control) individuals during a 280 d experiment in

running seawater

Nassarius reticulatus

Cyclope neritea

Control

Time (d)

Num

ber

of d

ead

sna

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Mar Ecol Prog Ser 276: 147–159, 2004

(Reznick & Ghalambor 2001, Lee 2002). In several cases,bottleneck effects have been correlated with rapidrates of evolution (Tsutsui et al. 2000, Lee 2002). Thelarge genetic diversity in neutral markers of Cyclopeneritea from Arcachon cannot be directly correlatedwith adaptative evolution. However, the studied popu-lation is likely to originate from individuals comingfrom different geographic origins, and may haveevolved under various environmental conditions; thiswould favour its adaptation to a wide range of environ-mental conditions and promote its long-term establish-ment and spread.

Mechanisms responsible for biological invasions arenot only linked to intrinsic characteristics of theinvaders, but also to interspecific interactions at thecommunity/ecosystem level. Both competitive interac-tion and parasitism are considered as critical factors forthe success of an invasion (Mooney & Cleland 2001,Keane & Crawley 2002). Non-indigenous species intro-duced in a new environment are expected to interferewith native species that exploit common food re-sources. We therefore hypothesized that Cyclope ner-itea, as a scavenger, would interact with the autochtho-nous dogwhelk Nassarius reticulatus which isprobably the commonest scavenger gastropod in shal-low marine waters of western Europe (Britton & Mor-ton 1994). The N. reticulatus complex actually in-cludes 2 closely related species, N. reticulatus (L.) andN. nitidus (Jeffreys) (Sanjuan et al. 1997); however, wefound only N. reticulatus in field sampling in ArcachonBay and used this species in competition experiments.A prerequisite to any study on interspecific competi-tion is that species encounter each other so they mustoccur in the same habitat. Both C. neritea and N. retic-ulatus are extremely eurythermic and euryhaline but,according to Mars (1950), N. reticulatus is slightly lesseuryhaline than C. neritea (15 to 39‰ vs. 6 to 42‰,respectively). Actually, C. neritea occurs everywherein Arcachon Bay, whereas N. reticulatus does notinhabit the most eastern parts of the bay where salinitymay fall well below 15‰ in winter, due to large inputsof freshwater from rivers and drainage trenches(Bachelet & Dauvin 1993). The zonation pattern foundin Arcachon Bay, i.e. –0.8 to +3.1 m for C. neritea and–5.2 to + 1.9 m for N. reticulatus, is in agreement withliterature data which indicate that C. neritea occurs inthe Atlantic mainly in the intertidal (Morton 1960,Sauriau 1989) while N. reticulatus extends from lowtide level to 36 m depth (Fretter & Graham 1962).Although both species may occur on a wide range ofsediments, C. neritea in Arcachon Bay seems to berestricted to sands with low organic content, whereasN. reticulatus generally thrives best in substrates withhigh organic content (Tallmark 1980). Therefore, itappears that the habitats of both gastropods partially

overlap in Arcachon Bay, with populations of both speciesbeing in sympatry in relatively clean medium andcoarse sands located at tidal levels between ca. –1 and+1 m, where some interspecific competition may beexpected.

Competition for food between benthic scavengershas seldom been demonstrated experimentally. To ourknowledge, this has been attempted only by Morton& Yuen (2000) who used a combination of methodssuch as the assessment of food preferences, speed tobait, consumption rates, and the observation of indi-vidual behaviour when species were feeding together.We tested neither different bait species nor consump-tion rates by the 2 nassariid species. Behaviouralobservations (not reported here) in aquaria showedthat up to 20 individuals of each species could befound aggregated on a single dead cockle withoutinterfering with each other, probably because bothspecies possess an extendible proboscis that allowsfeeding at a distance of 2 to 3 cm; similar observationswere made by Morton (1960) on Cyclope neritea andby Morton & Yuen (2000) on Nassarius festivus. In thepresent study, we chose the speed of individuals toreach carrion and begin feeding as a method toassess competition. Our laboratory experimentsclearly showed that, in static water, C. neritea movedtowards food faster than N. reticulatus, whatever thedistances (20, 40, or 60 cm) between the prey and thenassariids were. This trend changed in flow condi-tions, at least when the nassariids were at distances of20 and 40 cm from the prey, when both speciesmoved with a similar speed. These results were con-sistent with the spontaneous moving observed with-out prey, C. neritea being active in both static andmoving water, and N. reticulatus moving sponta-neously mainly in flow conditions. It appears that: (1)in still water experimental conditions, similar to thoseprevailing on intertidal sand flats with slowly runningwater at low tide (i.e. the habitat preferred by C. ner-itea), C. neritea was more active and reached its preyfaster than N. reticulatus, thus having a competitiveadvantage over the latter; (2) in flow conditions, simi-lar to those prevailing in subtidal levels (i.e. the habi-tat preferred by N. reticulatus), the activity of N.reticulatus increased compared to static conditionsand its efficiency to reach a prey became similar tothat of C. neritea. Nevertheless, even in low hydrody-namic conditions, competitive exclusion of N. reticula-tus by C. neritea is unlikely, due to the abundance ofbivalve carrion in the field, especially in shellfishareas such as Arcachon Bay. The fact that the 2 spe-cies basically have different substrate preferenceswould be an additional enhancing factor for theestablishment of C. neritea because it can occupy anopen niche in its new distribution area.

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The present study demonstrated that the nativeNassarius reticulatus was much more heavily para-sitized than the introduced Cyclope neritea. A similarresult was found in South Africa with the native musselPerna perna and the introduced mussel Mytilus gallo-provincialis (Calvo-Ugarteburu & McQuaid 1998a).The lack of infection in C. neritea could be due to anatural resistance to digenean parasites, as observedin the gastropod Crepidula fornicata in its native area(Pechenik et al. 2001). However, this is not the casebecause C. neritea in the Mediterranean can beheavily parasitized with the same digenean species asthose observed at Arcachon in N. reticulatus, e.g.Himasthla quissetensis (Prévot 1974). Therefore, it isdifficult to explain why C. neritea was not parasitizedin Arcachon Bay where both parasite and host species(final host: lariid birds; second intermediate host: cock-les) were all present. Two hypotheses can be proposed.The first one is a misidentification of the parasites.For example, is Himasthla quissetensis really thesame species that parasitizes C. neritea (Prévot 1974),N. reticulatus (Russell-Pinto 1993, Desclaux et al. 2002)and Ilyanassa obsoleta (Stunkard 1938)? If not, the lackof infection of C. neritea in the introduction site couldbe due to the absence of the different hosts of the par-asite cycle (the lariid birds and the cockles in theMediterranean belong to the same genus but not to thesame species). An alternative hypothesis in explainingthe lack of parasites in C. neritea is that populations ofthe introduced nassariid have acquired a higher resis-tance to digenean infections.

When an introduced species does not bring its nat-ural suite of enemies/parasites with it, it should experi-ence a type of release or ‘freedom’ in its new environ-ment. This release will be of a magnitude proportionalto the ecological importance of the natural enemies leftbehind (Torchin et al. 2002). In the case of a competi-tion between introduced and indigenous species, para-site release can be an important advantage (Calvo-Ugarteburu & McQuaid 1998b). Indeed, parasiticinfection is recognized as a stressful factor which maylower the resistance of the host and its ability to chal-lenge an unparasitized species. The experiment com-paring mortality rates of infected and non-infectedNassarius reticulatus emphasized the disadvantage ofbeing parasitized, as already suggested in a similarexperiment with the mudsnail Hydrobia ulvae (deMontaudouin et al. 2003).

By using a multidisciplinary approach, this paperinvestigated several features expected to promote thelong-term establishment of a recent invader that colo-nized Arcachon Bay 30 yr ago. Our results suggest thatthe combination of various factors, i.e. recurrent intro-duction of individuals originating from different envi-ronments, a significant competitive ability and the lack

of heavy parasitic load, explains the settlement ofCyclope neritea in Arcachon Bay. Whether these fac-tors can promote a greater invasion success in the nearfuture and the long-term establishment of this speciesalong the Atlantic French coast, although likely, is stillan opened issue. In particular, the role played byclimate change is still to be investigated since, asalready reported for marine benthic communities(Glémarec 1979) and invasive species such as C. ner-itea (Sauriau 1991) and Crassostrea gigas (Drinkwaard1999), climate warming is expected to favour popula-tion dynamics and further northwards expansion ofwarm-temperate species. Experimental studies andgenetic analysis of other French populations of C. neriteawould be useful to check for this effect as well as toverify the consistency of the patterns observed in theArcachon population.

Acknowledgements. This research was supported by the‘Invasions Biologiques (INVABIO)’ programme (project No.D4E/SRP/01115) of the Ministère de l’Ecologie et duDéveloppement Durable, France. F.V. received support forthe mtDNA analyses from the CNRS (ATIP programme) andthe Brittany Region (Programme ‘Accueil et Emergence’ andSequencing Platform–GenoMer/Genopole Ouest). C.D. andB.S.-B. were supported by a PhD grant from the Ministère dela Jeunesse, de l’Education Nationale et de la Recherche. Weacknowledge helpful comments from 2 anonymous referees.

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Editorial responsibility: Otto Kinne (Editor), Oldendorf/Luhe, Germany

Submitted: November 21, 2003; Accepted: April 22, 2004Proofs received from author(s): July 14, 2004